Concentrated Secretome of Adipose Stromal Cells Limits Influenza A Virus-Induced Lung Injury in Mice.

Despite vaccination and antivirals, influenza remains a communicable disease of high burden, with limited therapeutic options available to patients that develop complications. Here, we report the development and preclinical characterization of Adipose Stromal Cell (ASC) concentrated secretome (CS), generated by process adaptable to current Good Manufacturing Practices (cGMP) standards. We demonstrate that ASC-CS limits pulmonary histopathological changes, infiltration of inflammatory cells, protein leak, water accumulation, and arterial oxygen saturation (spO2) reduction in murine model of lung infection with influenza A virus (IAV) when first administered six days post-infection. The ability to limit lung injury is sustained in ASC-CS preparations stored at -80 °C for three years. Priming of the ASC with inflammatory factors TNFα and IFNγ enhances ASC-CS ability to suppress lung injury. IAV infection is associated with dramatic increases in programmed cell death ligand (PDL1) and angiopoietin 2 (Angpt2) levels. ASC-CS application significantly reduces both PDL1 and Angpt2 levels. Neutralization of PDL1 with anti-mouse PDL1 antibody starting Day6 onward effectively ablates lung PDL1, but only non-significantly reduces Angpt2 release. Most importantly, late-phase PDL1 neutralization results in negligible suppression of protein leakage and inflammatory cell infiltration, suggesting that suppression of PDL1 does not play a critical role in ASC-CS therapeutic effects.

Distinct Factors Secreted by Adipose Stromal Cells Protect the Endothelium From Barrier Dysfunction and Apoptosis.

We have shown previously that adipose stromal cell (ASC)-derived conditioned media (CM) limited lung injury, endothelial barrier dysfunction, and apoptosis. Here, we used endothelial hyperpermeability and apoptosis assays to investigate how concentration processes affect endothelium-directed bioactivity of ASC-CM and to gain information on the nature of bioactive factors. Comparison of ASC-CM concentrated with differential molecular weight (MW) cutoff filters showed that endothelial barrier protection depended on the species-specific factors in ASC-CM fractionated with MW > 50 kDa. Known barrier regulators-keratin growth factor (KGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF)-were detected in ASC-CM fraction of > 100 kDa. Pretreatment of endothelial monolayers with concentrations of KGF, VEGF, and HGF detected in ASC-CM showed that only KGF and HGF protect the endothelium from barrier dysfunction. Depletion of KGF and HGF from ASC-CM attenuated ASC-CM's ability to protect the endothelial barrier. In contrast to barrier-protective factors, apoptosis-protective factors fractionated with MW < 3 kDa and were not species-specific. Application of donors of apoptosis-mitigating gases showed that the CO donor carbon monoxide-releasing molecule 2 (CORM2) protected the endothelium from apoptosis, while the H2S donor NaSH did not. Knockdown of CO-generating heme oxygenase 1 in ASC attenuated ASC-CM's ability to protect the endothelium from apoptosis. We have shown that tumor necrosis factor alpha (TNFα)-induced apoptosis in endothelium is c-Jun N-terminal kinase (JNK)-dependent, and JNK activation is inhibited by ASC-CM pretreatment of endothelial cells. ASC-CM from heme oxygenase 1-depleted ASC displayed attenuated ability to suppress endothelial JNK activation, suggesting that CO-mediated protection of the endothelium from apoptosis is achieved by the downregulation of the JNK pathway. Altogether, our results demonstrate that the concentration of ASC-CM with low MW cutoff filters significantly reduces its anti-apoptotic activity while preserving its barrier-protective activity.

HIV-Nef Protein Persists in the Lungs of Aviremic Patients with HIV and Induces Endothelial Cell Death.

It remains a mystery why HIV-associated end-organ pathologies persist in the era of combined antiretroviral therapy (ART). One possible mechanism is the continued production of HIV-encoded proteins in latently HIV-infected T cells and macrophages. The proapoptotic protein HIV-Nef persists in the blood of ART-treated patients within extracellular vesicles (EVs) and peripheral blood mononuclear cells. Here we demonstrate that HIV-Nef is present in cells and EVs isolated from BAL of patients on ART. We hypothesize that HIV-Nef persistence in the lung induces endothelial apoptosis leading to endothelial dysfunction and further pulmonary vascular pathologies. The presence of HIV-Nef in patients with HIV correlates with the surface expression of the proapoptotic endothelial-monocyte-activating polypeptide II (EMAPII), which was implicated in progression of pulmonary emphysema via mechanisms involving endothelial cell death. HIV-Nef protein induces EMAPII surface expression in human embryonic kidney 293T cells, T cells, and human and mouse lung endothelial cells. HIV-Nef packages itself into EVs and increases the amount of EVs secreted from Nef-expressing T cells and Nef-transfected human embryonic kidney 293T cells. EVs from BAL of HIV+ patients and Nef-transfected cells induce apoptosis in lung microvascular endothelial cells by upregulating EMAPII surface expression in a PAK2-dependent fashion. Transgenic expression of HIV-Nef in vascular endothelial-cadherin+ endothelial cells leads to lung rarefaction, characterized by reduced alveoli and overall increase in lung inspiratory capacity. These changes occur concomitantly with lung endothelial cell apoptosis. Together, these data suggest that HIV-Nef induces endothelial cell apoptosis via an EMAPII-dependent mechanism that is sufficient to cause pulmonary vascular pathologies even in the absence of inflammation.

EMAPII Monoclonal Antibody Ameliorates Influenza A Virus-Induced Lung Injury.

Influenza A virus (IAV) remains a major worldwide health threat, especially to high-risk populations, including the young and elderly. There is an unmet clinical need for therapy that will protect the lungs from damage caused by lower respiratory infection. Here, we analyzed the role of EMAPII, a stress- and virus-induced pro-inflammatory and pro-apoptotic factor, in IAV-induced lung injury. First, we demonstrated that IAV induces EMAPII surface translocation, release, and apoptosis in cultured endothelial and epithelial cells. Next, we showed that IAV induces EMAPII surface translocation and release to bronchoalveolar lavage fluid (BALF) in mouse lungs, concomitant with increases in caspase 3 activity. Injection of monoclonal antibody (mAb) against EMAPII attenuated IAV-induced EMAPII levels, weight loss, reduction of blood oxygenation, lung edema, and increase of the pro-inflammatory cytokine TNF alpha. In accordance with the pro-apoptotic properties of EMAPII, levels of caspase 3 activity in BALF were also decreased by mAb treatment. Moreover, we detected EMAPII mAb-induced increase in lung levels of M2-like macrophage markers YM1 and CD206. All together, these data strongly suggest that EMAPII mAb ameliorates IAV-induced lung injury by limiting lung cell apoptosis and shifting the host inflammatory setting toward resolution of inflammation.

AMD3100 ameliorates cigarette smoke-induced emphysema-like manifestations in mice.

We have shown that cigarette smoke (CS)-induced pulmonary emphysema-like manifestations are preceded by marked suppression of the number and function of bone marrow hematopoietic progenitor cells (HPCs). To investigate whether a limited availability of HPCs may contribute to CS-induced lung injury, we used a Food and Drug Administration-approved antagonist of the interactions of stromal cell-derived factor 1 (SDF-1) with its chemokine receptor CXCR4 to promote intermittent HPC mobilization and tested its ability to limit emphysema-like injury following chronic CS. We administered AMD3100 (5mg/kg) to mice during a chronic CS exposure protocol of up to 24 wk. AMD3100 treatment did not affect either lung SDF-1 levels, which were reduced by CS, or lung inflammatory cell counts. However, AMD3100 markedly improved CS-induced bone marrow HPC suppression and significantly ameliorated emphysema-like end points, such as alveolar airspace size, lung volumes, and lung static compliance. These results suggest that antagonism of SDF-1 binding to CXCR4 is associated with protection of both bone marrow and lungs during chronic CS exposure, thus encouraging future studies of potential therapeutic benefit of AMD3100 in emphysema.

Pulmonary Retention of Adipose Stromal Cells Following Intravenous Delivery Is Markedly Altered in the Presence of ARDS.

Transplantation of mesenchymal stromal cells (MSCs) has been shown to effectively prevent lung injury in several preclinical models of acute respiratory distress syndrome (ARDS). Since MSC therapy is tested in clinical trials for ARDS, there is an increased need to define the dynamics of cell trafficking and organ-specific accumulation. We examined how the presence of ARDS changes retention and organ-specific distribution of intravenously delivered MSCs isolated from subcutaneous adipose tissue [adipose-derived stem cells (ADSCs)]. This type of cell therapy was previously shown to ameliorate ARDS pathology. ARDS was triggered by lipopolysaccharide (LPS) aspiration, 4 h after which 300,000 murine CRE+ ADSCs were delivered intravenously. The distribution of ADSCs in the lungs and other organs was assessed by real-time polymerase chain reaction (PCR) of genomic DNA. As anticipated, the majority of delivered ADSCs accumulated in the lungs of both control and LPS-challenged mice, with minor amounts distributed to the liver, kidney, spleen, heart, and brain. Interestingly, within 2 h following ADSC administration, LPS-challenged lungs retained significantly lower levels of ADSCs compared to control lungs (∼7% vs. 15% of the original dose, respectively), whereas the liver, kidney, spleen, and brain of ARDS-affected animals retained significantly higher numbers of ADSCs compared to control animals. In contrast, 48 h later, only LPS-challenged lungs continued to retain ADSCs (∼3% of the original dose), whereas the lungs of control animals and nonpulmonary organs in either control or ARDS mice had no detectable levels of ADSCs. Our data suggest that the pulmonary microenvironment during ARDS may lessen the pulmonary capillary occlusion by MSCs immediately following cell delivery while facilitating pulmonary retention of the cells.

Taxol alleviates 2-methoxyestradiol-induced endothelial permeability.

We have previously shown that the anti-cancer agent 2-methoxyestradiol (2ME) induces hyperpermeability across endothelial monolayers. Here, we show that both microtubule disruptor, 2ME, and microtubule stabilizer, paclitaxel (taxol), increase vascular lung permeability in vitro and in vivo. Simultaneous application of 2ME and taxol alleviates 2ME-induced endothelial barrier dysfunction, which is evident by the decreased Evans Blue Dye accumulation in lung tissue and increased transendothelial resistance across monolayers. 2ME significantly increases the level of p38 and MLC phosphorylation in both endothelial monolayers and murine lungs; this increase is suppressed in the presence of taxol. Taxol treatment leads to an immediate and sustained increase in tubulin acetylation in human pulmonary artery endothelial cells (HPAEC). Surprisingly, 2ME treatment also increases tubulin acetylation; however, the onset of this process is delayed and coincides with the stage of a partial barrier restoration in HPAEC monolayer. Inhibition of histone deacetylase 6 (HDAC6) with tubacin increases tubulin acetylation level, suppresses 2ME-induced HSP27 and MLC phosphorylation, and decreases 2ME-induced barrier dysfunction, suggesting barrier-protective and/or barrier-restorative role for tubulin acetylation in vascular endothelium.

Ezrin, radixin, and moesin are phosphorylated in response to 2-methoxyestradiol and modulate endothelial hyperpermeability.

We showed previously that microtubule disruptor 2-methoxyestradiol (2ME) induces hyperpermeability of the endothelial monolayer via mechanisms that include the activation of p38 and Rho kinase (ROCK) and rearrangement of the actin cytoskeleton. Using the protein kinase C (PKC) inhibitors Ro-31-7549 and Ro-32-0432, we show in vitro and in vivo that 2ME-induced barrier dysfunction is also PKC-dependent. The known PKC substrates ezrin, radixin, and moesin (ERM) were recently implicated in the regulation of endothelial permeability. This study tested the hypotheses that ERM proteins are phosphorylated in response to 2ME, and that this phosphorylation is involved in 2ME-induced barrier dysfunction. We show that the application of 2ME leads to a dramatic increase in the level of ERM phosphorylation. This increase is attenuated in cells pretreated with the microtubule stabilizer taxol. In human pulmonary artery endothelial cells (HPAECs), the phosphorylation of ERM occurs in a p38-dependent and PKC-dependent manner. The activation of p38 appears to occur upstream from the activation of PKC, in response to 2ME. Phosphorylated ERM are localized at the cell periphery during the early phase of response to 2ME (15 minutes), and colocalize with F-actin branching points during the later phase of response (60 minutes). Using the short interfering RNA approach, we also showed that individual ERM depletion significantly attenuates 2ME-induced hyperpermeability. HPAEC monolayers, depleted of ERM proteins and monolayers, overexpressing phosphorylation-deficient ERM mutants, exhibit less attenuation of 2ME-induced barrier disruption in response to the PKC inhibitor Ro-31-7549. These results suggest a critical role of PKC activation in response to microtubule-disrupting agents, and implicate the phosphorylation of ERM in the barrier dysfunction induced by 2ME.

The suppression of myosin light chain (MLC) phosphorylation during the response to lipopolysaccharide (LPS): beneficial or detrimental to endothelial barrier?

Sepsis-induced vascular leakage is a major underlying cause of the respiratory dysfunction seen in severe sepsis. Here, we studied the role of MLC phosphorylation in LPS-induced endothelial hyperpermeability and assessed how the changes in phospho-MLC distribution affect LPS-induced barrier dysfunction. We demonstrated that the changes in human lung microvascular endothelial permeability are preceded by the increase in intracellular calcium level, and increase in MYPT and MLC phosphorylation. Using the siRNA approach, we showed that both LPS-induced barrier dysfunction and MLC phosphorylation are attenuated by the depletion of the smooth muscle isoform of MLC kinase (MLCK) and Rho kinase 2 (ROCK2). Surprisingly, pharmacological inhibition of both ROCK1 and 2 with Y-27632 exacerbated LPS-induced drop in transendothelial resistance, although significantly decreasing MLC phosphorylation level. We next studied the involvement of protein kinase A (PKA)-dependent pathways in LPS-induced barrier dysfunction. We showed that LPS decreased the level of PKA-dependent phosphorylation in endothelial cells; and the pretreatment with forskolin or PKA activator bnz-cAMP counteracted this effect. Forskolin and bnz-cAMP also attenuated LPS-induced increase in MLC phosphorylation level. As we have shown earlier (Bogatcheva et al., 2009), forskolin and bnz-cAMP provide protection from LPS-induced barrier dysfunction. We compared the effects of bnz-cAMP and Y-27632 on phospho-MLC distribution and observed that while bnz-cAMP increased the association of the phospho-MLC signal with the cortical structures, Y-27632 decreased this association. These data indicate that an overall decrease in MLC phosphorylation could be either beneficial or detrimental to endothelial barrier, depending on the intracellular locale of major phospho-MLC changes.

Molecular mechanisms mediating protective effect of cAMP on lipopolysaccharide (LPS)-induced human lung microvascular endothelial cells (HLMVEC) hyperpermeability.

Up to date, the nature of the sepsis-induced vascular leakage is understood only partially, which limits pharmacological approaches for its management. Here we studied the protective effect of cAMP using endotoxin-induced hyperpermeability as a model for barrier dysfunction observed in gram-negative sepsis. We demonstrated that the alleviation of lipopolysaccharide (LPS)-induced barrier compromise could be achieved by the specific activation of either protein kinase A (PKA) or Epac with cAMP analogs Bnz-cAMP or O-Me-cAMP, respectively. We next studied the involvement of PKA substrates VASP and filamin1 in barrier maintenance and LPS-induced barrier compromise. Depletion of both VASP and filamin1 with the specific siRNAs significantly exacerbated both the quiescent cells barrier and LPS-induced barrier dysfunction, suggesting barrier-protective role of these proteins. VASP depletion was associated with the more severe loss of ZO-1 peripheral staining in response to LPS, whereas filamin1-depleted cells reacted to LPS with more robust stress fiber induction and more profound changes in ZO-1 and VE-cadherin peripheral organization. Both VASP and filamin1 phosphorylation was significantly increased as a result of PKA activation. We next analyzed the effect of VASP and filamin1 depletion on the PKA-dependent alleviation of LPS-induced barrier compromise. We observed that Bnz-cAMP ability to counteract LPS-induced hyperpermeability was attenuated only by VASP, but not filamin1 depletion. Our data indicate that while PKA-dependent VASP phosphorylation contributes to the protective effect of cAMP elicited on LPS-compromised monolayers, filamin1 phosphorylation is unlikely to play a significant role in this process.

TIMAP is a positive regulator of pulmonary endothelial barrier function.

TGF-beta-inhibited membrane-associated protein, TIMAP, is expressed at high levels in endothelial cells (EC). It is regarded as a member of the MYPT (myosin phosphatase target subunit) family of protein phosphatase 1 (PP1) regulatory subunits; however, its function in EC is not clear. In our pull-down experiments, recombinant TIMAP binds preferentially the beta-isoform of the catalytic subunit of PP1 (PP1cbeta) from pulmonary artery EC. As PP1cbeta, but not PP1calpha, binds with MYPT1 into functional complex, these results suggest that TIMAP is a novel regulatory subunit of myosin phosphatase in EC. TIMAP depletion by small interfering RNA (siRNA) technique attenuates increases in transendothelial electrical resistance induced by EC barrier-protective agents (sphingosine-1-phosphate, ATP) and enhances the effect of barrier-compromising agents (thrombin, nocodazole) demonstrating a barrier-protective role of TIMAP in EC. Immunofluorescent staining revealed colocalization of TIMAP with membrane/cytoskeletal protein, moesin. Moreover, TIMAP coimmunoprecipitates with moesin suggesting the involvement of TIMAP/moesin interaction in TIMAP-mediated EC barrier enhancement. Activation of cAMP/PKA cascade by forskolin, which has a barrier-protective effect against thrombin-induced EC permeability, attenuates thrombin-induced phosphorylation of moesin at the cell periphery of control siRNA-treated EC. On the contrary, in TIMAP-depleted EC, forskolin failed to affect the level of moesin phosphorylation at the cell edges. These results suggest the involvement of TIMAP in PKA-mediated moesin dephosphorylation and the importance of this dephosphorylation in TIMAP-mediated EC barrier protection.

Relaxin promotes prostate cancer progression.

PURPOSE: To understand the role of relaxin peptide in prostate cancer, we analyzed the expression of relaxin and its receptor in human prostate cancer samples, the effects of relaxin signaling on cancer cell phenotype in vitro, and the effects of increased serum relaxin concentrations on cancer progression in vivo. EXPERIMENTAL DESIGN: The relaxin and its receptor leucine-rich repeat containing G protein-coupled receptor 7 (LGR7) expression were studied by quantitative reverse transcription-PCR (11 benign and 44 cancer tissue samples) and by relaxin immunohistochemistry using tissue microarrays containing 10 normal and 69 cancer samples. The effects of relaxin treatment and endogenous relaxin/LGR7 suppression via short interfering RNA in PC-3 and LNCaP cells were analyzed in vitro. The effect of transgenic relaxin overexpression [Tg(Rln1)] on cancer growth and survival was evaluated in autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP). RESULTS: The relaxin mRNA expression was significantly higher in recurrent prostate cancer samples. In tissue microarrays of the 10 normal tissues, 8 had low staining in epithelial cells, whereas only 1 of 9 high-grade prostatic intraepithelial neoplasia lesions had low expression (P = 0.005) and only 29 of 65 cancers had low expression (P = 0.047). Stimulation with relaxin increased cell proliferation, invasiveness, and adhesion in vitro. The suppression of relaxin/LGR7 via short interfering RNAs decreased cell invasiveness by 90% to 95% and growth by 10% to 25% and increased cell apoptosis 0.6 to 2.2 times. The Tg(Rln1) TRAMP males had shorter median survival time, associated with the decreased apoptosis of tumor cells, compared with non-Tg(Rln1) TRAMP animals. CONCLUSIONS: Relaxin signaling plays a role in prostate cancer progression.

Involvement of microtubules, p38, and Rho kinases pathway in 2-methoxyestradiol-induced lung vascular barrier dysfunction.

2-Methoxyestradiol (2ME), a promising anti-tumor agent, is currently tested in phase I/II clinical trial to assess drug tolerance and clinical effects. 2ME is known to affect microtubule (MT) polymerization rather than act through estrogen receptors. We hypothesized that 2ME, similar to other MT inhibitors, disrupts endothelial barrier properties. We show that 2ME decreases transendothelial electrical resistance and increases FITC-dextran leakage across human pulmonary artery endothelial monolayer, which correlates with 2ME-induced MT depolymerization. Pretreatment of endothelium with MT stabilizer taxol significantly attenuates the decrease in transendothelial resistance. 2ME treatment results in the induction of F-actin stress fibers, accompanied by the increase in myosin light chain (MLC) phosphorylation. The experiments with Rho kinase (ROCK) and MLC kinase inhibitors and ROCK small interfering RNA (siRNA) revealed that increase in MLC phosphorylation is attributed to the ROCK activation rather than MLC kinase activation. 2ME induces significant ERK1/2, p38, and JNK phosphorylation and activation; however, only p38 activation is relevant to the 2ME-induced endothelial hyperpermeability. p38 activation is accompanied by a marked increase in MAPKAP2 and 27-kDa heat shock protein (HSP27) phosphorylation level. Taxol significantly decreases p38 phosphorylation and activation in response to 2ME stimulation. Vice versa, p38 inhibitor SB203580 attenuates MT rearrangement in 2ME-challenged cells. Together, these results indicate that 2ME-induced barrier disruption is governed by MT depolymerization and p38- and ROCK-dependent mechanisms. The fact that certain concentrations of 2ME induce endothelial hyperpermeability suggests that the issue of the maximum-tolerated dose of 2ME for cancer treatment should be addressed with caution.

The role of relaxin in endometrial cancer.

Relaxin (RLN) is a naturally occurring hormone that is known to modulate connective tissue remodeling in the uterus and cervix. Our goal was to investigate the role of RLN in endometrial cancer. RLN expression was evaluated using immunohistochemistry in 57 samples of invasive endometrial carcinoma (EC) and ten benign endometrial tissues. 67% of high-stage (III/IV) tumors demonstrated strong RLN expression compared to 37% of low-stage (I/II) cases. Strong RLN expression associated significantly with high-grade and depth of myometrial invasion. Notably, strong RLN expression was associated with a significantly shorter overall survival (p < 0.005) compared to weak or moderate expression. Using RT-PCR, the expression of RLN and its receptor (LGR7) was detected in EC cell lines (HEC-1B and KLE); in addition, LGR7 was expressed in 86% of 15 primary EC tissue samples. Exogenous RLN stimulation caused a significant increase in migration and invasion in both cell lines, but did not stimulate proliferation in vitro. Addition of the MMP inhibitor, FN439 abolished the stimulatory effect of RLN on invasion in both HEC-1B and KLE cells. RLN stimulation caused a significant increase in levels of activated MMP-2 in KLE cells and activated MMP-9 in HEC-1B cells compared to unstimulated cells. Inhibition of endogenous RLN signaling via siRNA targeted to LGR7 caused a significant reduction of EC cell invasiveness. Our results indicate that RLN overexpression is significantly asso- ciated with aggressive features such as high-grade and deep myometrial invasion. We provide the first evidence that overexpression of RLN is associated with poor clinical outcome in women with EC. RLN stimulation enhances the invasive potential of endometrial cancer cells by upregulating MMPs. In turn, downregulation of endogenous RLN signaling decreases invasiveness of endometrial cancer cells. These novel findings may have therapeutic implications in the management of patients with endometrial carcinoma.

Mitogen-activated protein kinases in endothelial pathophysiology.

Endothelial cells continuously respond to extracellular stimuli such as chemical signals produced by circulating blood elements or mechanical forces such as shear stress. Proinflammatory cytokines, mitogens, reactive oxygen species, and shear stress trigger signal molecules to initiate multiple intracellular pathways, which often converge at mitogen-activated protein (MAP) kinase activation. The MAP kinase superfamily represents a burgeoning area of clinical investigation for treatment of various inflammatory and oncologic diseases and plays an essential role in mediating response to infection, ischemia/reperfusion injury, and vessel healing and remodeling through regulation of such diverse phenomena as endothelial cell proliferation, migration, apoptosis, and endothelial barrier function. The downstream effects of MAP kinase activation include modulation of gene expression via up-regulation of various transcription factors. In addition to these sustained effects, MAP kinases coordinate more immediate responses that affect dynamic cytoskeletal rearrangements necessary for cell migration and regulation of barrier function. This review discusses the important regulatory roles of MAP kinases in the vital physiologic functions of endothelium, focusing mainly on the role of MAP kinases in the maintenance of endothelial barrier.

Mutations of the GREAT gene cause cryptorchidism.

In humans, failure of testicular descent (cryptorchidism) is one of the most frequent congenital malformations, affecting 1-3% of newborn boys. The clinical consequences of this abnormality are infertility in adulthood and a significantly increased risk of testicular malignancy. Recently, we described a mouse transgene insertional mutation, crsp, causing high intraabdominal cryptorchidism in homozygous males. A candidate gene Great (G-protein-coupled receptor affecting testis descent), was identified within the transgene integration site. Great encodes a seven-transmembrane receptor with a close similarity to the glycoprotein hormone receptors. The Great gene is highly expressed in the gubernaculum, the ligament that controls testicular movement during development, and therefore may be responsible for mediating hormonal signals that affect testicular descent. Here we show that genetic targeting of the Great gene in mice causes infertile bilateral intraabdominal cryptorchidism. The mutant gubernaculae fail to differentiate, indicating that the Great gene controls their development. Mutation screening of the human GREAT gene was performed using DHPLC analysis of the genomic DNA from 60 cryptorchid patients. Nucleotide variations in GREAT cDNA were found in both the patient and the control populations. A unique missense mutation (T222P) in the ectodomain of the GREAT receptor was identified in one of the patients. This mutant receptor fails to respond to ligand stimulation, implicating the GREAT gene in the etiology in some cases of cryptorchidism in humans.